ObjectiveTo determine cerebrospinal fluid levels of osteopontin (OPN), a proinflammatory cytokine that was found to be overexpressed in multiple sclerosis lesions and increased in plasma during relapses and in secondary progressive multiple sclerosis.

ConclusionOsteopontin in the cerebrospinal fluid may be, in part, of central nervous system origin, and may play an important role in central nervous system inflammation.

Proinflammatory cytokines and T cells reactive against myelin proteins play an important role in the pathogenesis of multiple sclerosis (MS) and its experimental model, experimental autoimmune encephalomyelitis (EAE).1,2

Osteopontin is highly upregulated in the brains of persons with MS,5,6 and in the spinal cords of mice with EAE.5 In OPN-deficient mice, EAE shows a greater number of remissions, less progression, and higher IL-10 levels.5 Moreover, OPN induces relapses and progression of EAE.4

Osteopontin levels are higher in the serum of MS patients during relapse than during remission; however, no differences in OPN levels were found between patients with relapsing-remitting MS and control group participants.7,8 Plasma OPN levels are higher in patients with secondary progressive MS than in healthy controls.9

We measured OPN levels in the cerebrospinal fluid (CSF) of MS patients and compared them with the levels in patients with other inflammatory and noninflammatory neurological diseases.

Cytokine levels were compared between groups using the Mann-Whitney U test. Correlations were explored using the Pearson coefficient.

RESULTS

The mean (SD) levels of OPN in plasma were 380 (236) ng/mL in the MS group, 386 (271) ng/mL in the OIND group, and 377 (121) ng/mL in the NIND group. Differences were not statistically significant. In the MS patients, the mean (SD) level of disability, using Kurtzke's expanded disability status scale, was 2.8 (1.6) (range, 1-6.5).

In the relapsing-remitting MS group, the mean (SD) interval between the last clinical relapse and sample collection was 5.8 (7) months.

Multiple sclerosis patients had a mean (SD) CSF OPN level of 415 (186) ng/mL, similar to OIND patients (563 [411] ng/mL) (difference not statistically significant; P = .5). Both were higher than those of the NIND patients (286 [150] ng/mL) (P = .02 for patients with MS; P = .02 for patients with OIND) (Figure). Patients with MS and patients with OIND together had higher OPN levels than the NIND control group (457 [271] ng/mL vs 289 [146] ng/mL; P = .008).

Cerebrospinal fluid IL-10 was detectable in 15 patients (mean [SD], 3.6 [2.9] pg/mL; range, 0.02-7.9 pg/mL) of whom 8 had MS (3 with simultaneously detectable IL-12p40; all with high IL-12p40 and low IL-10). Osteopontin levels did not correlate with either IL-10 levels or IL-10 detection. Levels of MMP-9 were below theELISA detection limit in all samples.

There was no correlation between OPN levels in CSF and plasma. Levels of CSF OPN appeared consistently higher than plasma levels, although differences were not statistically significant (P = .1). There was no correlation between the CSF OPN levels and MS type, though with 1 secondary progressive MS patient, analysis for secondary progressive MS was not possible. There was no correlation between OPN levels and Kurtzke's expanded disability status scale or the interval since the last relapse.

COMMENT

We found significantly elevated levels of OPN in the CSF of patients with MS and OIND compared with those with NIND. Levels of OPN were slightly higher in MS CSF than in plasma, suggesting a component of CSF OPN was contributed to by central nervous system (CNS)–derived OPN. This is consistent with the ability of CNS cells to express OPN,11 and with the finding of high OPN levels in MS patients' brains.5

The fact that OPN levels were increased in the CSF of relapsing-remitting MS patients who were in clinical remission suggests continuous upregulation of OPN in the CNS of patients with MS, regardless of clinical disease activity. The same pattern of upregulation was found for MMP-9 in the CSF of patients with MS, suggesting the destructive proteolytic process is continuous.12 Interestingly, OPN activates pro–MMP-913 and its conversion to an active enzyme. Therefore, the similar patterns in the CSF of patients with MS may be explained by OPN activating the destructive effects of MMP-9. This hypothesis remains plausible despite the fact that, in our samples, levels of MMP-9 were below the ELISA detection limit. In a number of CSF samples from patients of Leppert et al,12 MMP-9 was undetectable by ELISA but detectable by zymography; however, we had insufficient CSF to conduct zymographies.

Osteopontin was increased in the CSF of OIND patients, indicating that OPN upregulation is not MS-specific but is inflammation-specific. The higher levels seen in CSF compared with plasma also suggest a contribution from CNS OPN. In these studies, we did not have the opportunity to compare CSF OPN levels between clinical relapse and remission. However, although previous studies showed no significant difference in plasma OPN levels between MS and healthy participants,7 we detected differences in CSF OPN levels between stable MS patients and participants in the control group. The majority of our patients had a mild to moderate disability; this, together with CSF OPN being increased regardless of the clinical disease activity, further suggests that OPN contributes continuously, and possibly from early stages, to MS pathology.

Osteopontin has been implicated in remyelination after experimental demyelination. Osteopontin was significantly upregulated in both the demyelination and re myelinating phases, suggesting a dual role.15 A positive OPN effect in remyelination was suggested by its ability to stimulate oligodendrocytes.15 Whether this dual OPN role is also played in MS, where demyelinating and remyelinating lesions coexist, remains to be established. Our data, including the correlation of CSF OPN levels with the detection of proinflammatory IL-12p40, but not of antiinflammatory IL-10, and recent observations in EAE,3 however, strongly suggest that in CNS inflammatory demyelination, OPN net effect is detrimental.

In conclusion, we found elevated OPN levels in the CSF of patients with MS and OIND which tend to exceed plasma levels, suggesting that the sources of OPN in these conditions include a contribution from CNS resident or infiltrating cells.